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Firefly Luciferase Complementation Imaging Assay for Protein-Protein Interactions in
Plants
Huamin Chen1, 2, Yan Zou2, Yulei Shang2, Huiqiong Lin2, Yujing Wang2, Run Cai1, Xiaoyan Tang3,
and Jian-Min Zhou2*
1 School of Agriculture and Biology, Shanghai Jiaotong University
2 National Institute of Biological Sciences, Beijing
3 Department of Plant Pathology, Kansas State University
* Corresponding author; e-mail zhoujianmin@nibs.ac.cn. fax 86-10-80726687.
Plant Physiology Preview. Published on December 7, 2007, as DOI:10.1104/pp.107.111740
Copyright 2007 by the American Society of Plant Biologists
Chen et al., page 2
Running title: Luciferase complementation for protein-protein interactions
This work was supported by a grant from Chinese Ministry of Science and Technology
(2003-AA210090).
Corresponding author: Jian-Min Zhou; e-mail zhoujianmin@nibs.ac.cn. fax 86-10-80726687.
The author responsible for distribution of materials integral to the findings presented in this article
in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Jian-Min Zhou (zhoujianmin@nibs.ac.cn).
RESEARCH CATEGORY: Breakthrough Technologies
Chen et al., page 3
ABSTRACT
The development of sensitive and versatile techniques to detect protein-protein interactions in vivo
is important for understanding protein functions. The previously described techniques
Fluorescence Resonance Energy Transfer (FRET) and Bimolecular Fluorescence
Complementation (BiFC) that are used widely for protein-protein interaction studies in plants
require extensive instrumentation. To facilitate protein-protein interactions studies in plants, we
adopted the luciferase complementation imaging (LCI) assay. The amino-terminal and
carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when
fused to two interacting proteins, and that can be visualized with a low light imaging system. A
series of plasmid constructs were made to enable the transient expression of fusion proteins or
generation of stable transgenic plants. We tested 9 pairs of proteins known to interact in plants,
including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant,
proteins of the SCF E3 ligase complex, the HSP90 chaperone complex, components of disease
resistance protein complex, and transcription factors. In each case, strong luciferase
complementation was observed for positive interactions. Mutants that are known to compromise
protein-protein interactions showed little or much reduced luciferase activity. Thus the assay is
simple, reliable, and quantitative in detection of protein-protein interactions in plants.
Chen et al., page 4
Noncovalent interactions among proteins are vital for all aspects of cellular processes. Thus the
identification and characterization of interacting proteins are key to our understanding of protein
functions. A plethora of techniques have been developed to detect protein-protein interactions in
vitro and in vivo (Piehler, 2005). The most widely used among these techniques is the yeast
two-hybrid assay which is ideal for large scale screening for interacting proteins and the
construction of protein interactomes (Fields and Song, 1989; Li et al., 2004). However, the yeast
two-hybrid assay detects protein-protein interactions under heterologous conditions, and results
must be validated by assays under physiological conditions. Examination of protein-protein
interactions under physiological conditions is often technically demanding and requires tedious
procedures. For example, the co-immunoprecipitation assay requires specific antibodies, lengthy
procedures that are influenced by parameters such as schemes for protein extraction, binding and
washing, and expertise of individuals performing the experiment. Thus, the results are often
variable from laboratory to laboratory. Tandem affinity purification (TAP) represents a more
advanced technique primarily designed to identify new proteins in a protein complex in a native
state (Puig et al., 2001; Rohila et al., 2006).
The development of reporter-based in vivo protein-protein interaction assays such as Fluorescence
Resonance Energy Transfer (FRET; Heim and Tsien, 1996; Ha et al., 1996; Mahajan et al., 1998),
the related technology Bioluminescence Resonance Energy Transfer (BRET; Xu et al., 1999;
Subramanian et al., 2006), and Bimolecular Fluorescence Complementation (BiFC; Hu et al.,
2002) assays has significantly advanced the measurement of protein-protein interactions in vivo.
These assays are instrumental for a number of important discoveries in mammalian studies. The
Chen et al., page 5
application of FRET and BRET in plant biology, however, has encountered significant difficulties
despite of sporadic successes (Shen et al., 2007). Both assays require sophisticated microscopy
and computation. BiFC is relatively simple compared to FRET and BRET and has been used in a
number of plant protein-protein interaction studies (Walter et al., 2004; Bracha-Drori et al., 2004;
Dong et al., 2006; Quan et al., 2007). FRET and BiFC are technically challenging when a large
number of protein pairs are to be tested. Furthermore, the application of FRET and BiFC assays in
plants is complicated by the autofluorescence generated by cell wall, chloroplast and other cell
structures. Finally, photobleaching and phototoxicity caused by the external light source for
excitation of fluorescence also restrict the application of the reporter-based assays in plants (Dixit
et al., 2006).
Alternative reporter-based methods for protein-protein interactions have been developed using
protein fragment complementation coupled with enzymatic assays. For example, expression of
β-galactosidase fragments fused to interacting proteins reconstitutes the enzymatic activity in E.
coli (Rossi et al., 1997). Similarly, 1-β-lactamase has been used to detect protein-protein
interactions in mammalian cells (Galarneau et al., 2002). Protein fragment complementation based
on the reconstitution of murine dihydrofolate reductase (Remy and Michnick, 1999) was used to
detect NPR1-TGA2 interaction in plants (Subramaniam et al., 2001). These assays typically
require the addition of fluorescence-generating substrates and thus also suffer from pitfalls of
FRET and BiFC. Recently, an improved firefly luciferase complementation imaging (LCI) assay
was developed for protein-protein interactions in animals (Luker et al., 2004). The firefly
luciferase enzyme is divided into the N- and C-terminal halves that do not spontaneously
Chen et al., page 6
reassemble and function. Luciferase activity occurs only when the two fused proteins interact,
resulting in reconstituted luciferase enzyme, which can be detected by luminometer or a low light
imaging device. The assay measures dynamic changes in protein-protein interactions and can be
used for both cell culture and whole animals. Because the luminescence was measured in the dark
and is not affected by autofluorescence, LCI is particularly attractive for plant studies. A very
recent report successfully used Renilla reniforms luciferase complementation assay to detect
interactions of two pairs of plant proteins in protoplasts (Fujikawa and Kato, 2007). The utility of
the firefly luciferase complementation imaging in plant protein-protein interaction studies remains
to be tested.
In this study, we developed a series of constructs and comprehensively tested the utility of firefly
luciferase-based LCI in plants. Tests with 9 pairs of proteins that are known to interact with
different strength in the plant cell showed that the firefly luciferase-based LCI assay is suitable for
detecting protein-protein interactions in both protoplasts and intact leaves. The assay is simple,
quantitative, highly sensitive, and can be used for transient expression or stable transgenic
expression of the interacting proteins. The system provides a new tool for plant protein-protein
interaction studies.
RESULTS
Constructs for LCI Assays
The firefly luciferase fragments 2-416 (NLuc) and 398-550 (CLuc) were successfully used for
Chen et al., page 7
protein-protein interaction assays in the mammalian system (Luker et al., 2004). These two
fragments roughly correspond to the independently folded N-terminal and C-terminal domains that
are linked by a disordered flexible region (Conti et al., 1996). To test the utility of LCI in plants,
the NLuc and CLuc fragments were inserted into an expression cassette between the CaMV 35S
promoter and NOS terminator to form 35S::NLuc and 35S::CLuc, respectively (Figure 1). A
Gly/Ser linker between the LUC fragments and the multiple cloning sites (Luker et al., 2004) was
retained in the constructs to allow molecular mobility at the junction of the fusion proteins. Two
sets of constructs were made for LCI assays. The first set was made in a pUC19-based plasmid
designed for transfection of protoplasts or particle bombardment into plant tissues. The second set
was produced in pCAMBIA-based plasmid for generation of stable transgenic plants or
Agrobacterium-mediated transient expression. Multiple cloning sites were inserted N-terminus to
the NLuc fragment and C-terminus to the CLuc fragment. We selected 9 pairs of proteins that are
known to interact with different strength and possess a range of biochemical functions in the plant
cell.
Interaction between Bacterial Effectors and Host Proteins
Bacterial pathogens inject effector proteins into the host cells to regulate host
susceptibility/resistance to the bacterium (Nomura et al., 2006; Chisholm et al., 2006). We
previously showed that the Pseudomonas syringae effector protein AvrB targets Arabidopsis
protein RAR1 to promote virulence (Shang et al., 2006). To test if such an interaction can be
detected with the LCI assay, CLuc-AvrB was co-expressed with RAR1-NLuc in protoplasts. For
negative controls, we included SCaBP8 that functions in salinity tolerance (Quan et al., 2007). As
Chen et al., page 8
shown in Figure 2A, CLuc-AvrB and RAR1-NLuc co-expression led to strong luciferase activity
in the protoplasts that can be readily detected with a low light imaging system after the addition of
luciferin, the substrate for firefly luciferase. In contrast, RAR1-NLuc co-expressed with SCaBP8
construct showed only background level luciferase activity. The rar1-29 mutant carries a single
amino acid substitution that specifically disrupts its interaction with SGT1b (Shang et al., 2006).
This mutant showed normal interaction with AvrB (Figure 2A). To determine if the observed
luciferase activity was caused by different levels of proteins expressed in the protoplasts, we
examined respective NLuc and CLuc fusion proteins by western blot. The CLuc-SCaBP8 protein
was expressed at a level similar to CLuc-AvrB, and RAR1-NLuc protein was expressed at a
similar level in all samples. The results indicate that the strong luciferase activity was not caused
by higher levels of CLuc-AvrB and RAR1-NLuc proteins expressed in the cell but resulted from a
specific interaction between RAR1 and AvrB.
The P. syringae effector AvrPto interacts with the tomato serine/threonine protein kinase Pto (Tang
et al., 1996), the latter subsequently triggers resistance through the association with the
N-terminus of the resistance protein Prf (NPrf) but not the C-terminus of Prf (CPrf; Mucyn et al.,
2006). The interaction between AvrPto and Pto, however, has never been demonstrated in vivo. We
tested if LCI can be used to detect such an interaction. Figure 2B shows that co-expression of
Pto-NLuc with CLuc-NPrf, but not CLuc-SCaBP8 or CLuc-CPrf, resulted strong LUC activity.
CLuc-NPrf was accumulated to a level ~8 fold higher than CLuc-CPrf. However, the reconstituted
luciferase activity of Pto-NPrf combination was ~60 fold greater than the Pto-CPrf combination.
The results indicate that the Pto-NPrf interaction resulted in significant increase in luciferase
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activity, confirming previous co-immunoprecipitation results (Mucyn et al., 2006). Similarly,
co-expression of CLuc-AvrPto with Pto-Nluc in Arabidopsis protoplasts resulted in strong
complementation of luciferase activity. We recently showed that AvrPtoY89 makes direct contact
with Pto, and the AvrPtoY89D mutation abolishes the interaction in vitro (Xing et al., 2007).
Co-expression of CLuc- AvrPtoY89D with Pto-NLuc failed to show luciferase complementation
although the mutant and wild-type CLuc-AvrPto proteins accumulated to the same level in the
plant cell (Figure 2C), indicating that the interaction detected by LCI is highly specific.
Interaction of the HSP90 Complex Components
The HSP90 protein complex plays an important role in plant innate immunity. HSP90 and its
co-chaperones, SGT1 and RAR1, interact with each other, and all three components are required
for disease resistance. Arabidopsis contains two SGT1 genes, SGT1a and SGT1b, both functions in
stabilizing disease resistance proteins (Shirasu et al., 1999; Azevedo et al., 2002; Takahashi et al.,
2003). The RAR1 protein contains a CHORD I domain (a cysteine and histidine-rich domain), a
CC domain (a central cysteine-rich domain), and a CHORD II domain. CHORD I is required for
interaction with HSP90, whereas the CHORD II domain is required for interaction with SGT1. We
showed previously that the rar1-29 allele was compromised in the interaction with SGT1b in the
yeast two-hybrid assay (Shang et al., 2006). Figure 3A shows that the LCI assay in protoplasts
detected a specific interaction of RAR1 with SGT1b, but not ScaBP8. The rar1-29 mutant protein
accumulated to the similar level as the wild-type RAR1, but displayed much weaker interaction
with SGT1b. Similarly we tested the interaction of RAR1 with SGT1a and HSP90. As shown in
Figure 3B and 3C, the full-length RAR1 was capable of interacting with both SGT1a and HSP90.
Chen et al., page 10
The CLuc-CHORD I and CLuc-CHORD II domain fusion proteins accumulated to a similar level
as the full-length CLuc-RAR1 protein, but showed only a background level luciferase activity
when co-expressed with SGT1a-NLuc and HSP90-NLuc, respectively. These results are consistent
with the respective roles of CHORD I and CHORD II domains in the HSP90 complex.
Protein-Protein Interactions between WRKY Proteins
Transcription factors WRKY18, WRKY40, and WRKY60 play an important role in regulating
plant immunity. Interestingly, these transcription factors are able to form homo- or heterodimers,
and this interaction is the basis for a complex regulation of down-stream gene expression (Xu et
al., 2006). We tested the utility of LCI for the interaction between WRKY40 and WRKY18. As
shown in Figure 4, co-expression of CLuc-WRKY18 and WRKY40-NLuc in protoplasts strongly
complemented the luciferase activity compared to the negative control protoplasts co-expressing
CLuc-SCaBP8 and WRKY40-NLuc. The leucine zipper motif of these WRKYs is required for the
dimerization. Deletion of this motif (CLuc-WRKY18D) significantly reduced the luciferase
complementation with WRKY40-NLuc, even though 2-3 fold more CLuc-WRKY18D protein was
expressed in these protoplasts. These results indicate that the LCI assay is also useful for studying
interactions among transcription factors.
Interactions between SCF E3 Ubiquitin Ligase Complex Components
The SCF (SKP1-Cullin-F-box protein) complex is an E3 ubiquitin ligase regulating 26S
proteasome-dependent degradation of a variety of proteins and is central to plant development and
responses to the environment (Callis and Vierstra, 2000). SKP1 directly interacts with the F-box
Chen et al., page 11
proteins, the latter serve to recruit specific substrate proteins for degradation. F-box proteins form
a super family with members such as COI1 that regulates jasmonate signaling and EBF1 and
EBF2 that regulate ethylene signaling (Xu et al., 2002; Guo and Ecker, 2003; Potuschak et al.,
2003). In Arabidopsis, SKP1 is encoded by ASK1 and ASK2. Both ASK1 and ASK2 interact with
COI1 to regulate jasmonate signaling (Xu et al., 2002). We tested if LCI could be used to detect
the ASK1-COI1 interaction. Co-expression of CLuc-COI1 and ASK1-NLuc in protoplasts resulted
in strong luciferase activity (Figure 5). Co-transfection of an empty CLuc plasmid with
ASK1-NLuc showed a much weaker activity that was approximately 14% of protoplasts
co-expressing CLuc-COI1 and ASK1-NLuc, suggesting a specific interaction between ASK1 and
COI1 in plant cells.
EBF1 and EBF2 directly interact with their substrate protein EIN3 (Guo and Ecker, 2003;
Potuschak et al., 2003), a transcription factor, to regulate gene expression. Because this interaction
leads to the degradation of EIN3, such an interaction in vivo remains to be demonstrated. To test if
LCI was capable of detecting EBF1-EIN3 interaction in the plant cell, we used a truncated EBF1
containing the leucine-rich repeat (LRR) domain required for substrate-binding but lacking the
F-box domain. Co-expression of the CLuc-EBF1 with EIN3-NLuc resulted in strong luciferase
activity in protoplasts, whereas the co-expression of CLuc-EBF1 with SCaBP-NLuc showed only
background level activity, indicating that the interaction of an F-box protein with its substrate can
be successfully detected by LCI (Figure S1). Although the EBF1-NLuc fusion protein was not
detected by western blot, preventing a quantitative assessment of the protein-protein interaction,
the results are nevertheless consistent with previous yeast two-hybrid data (Guo and Ecker, 2003;
Chen et al., page 12
Potuschak et al., 2003).
Comparison of Reconstituted and Full-Length Firefly Luciferase Activity
We compared protoplasts co-expressing SGT1a-NLuc and CLuc-RAR1 with those transfected
with a 35S::LUC (full-length) construct (Figure S2). The latter showed ~30 fold stronger
luminescence. The simple calculation based on cell number would be such that the reconstituted
luciferase possesses ~3% of the native luciferease activity. However, the SGT1a-NLuc
accumulated to only ~10% of the full-length luciferase protein, whereas the CLuc-RAR1 protein
was accumulated to a level similar to the full-length luciferase. Because the expression of
CLuc-RAR1 alone never resulted in significant luminescence in numerous tests (less than 5
counts), the vast majority of CLuc-RAR1 is unlikely to function in the absence of SGT1a-NLuc
(Luker et al., 2004). Therefore, our adjusted estimate of the reconstituted luciferase activity is
~30% of that of the full-length protein.
Agrobacterium-Mediated Transient Expression for LCI Assays
Agrobacterium-mediated transient expression in Nicotiana benthamiana provides a convenient
system for the rapid analysis of protein functions in plants. We therefore tested if
Agrobacterium-mediated transient expression could be adopted for the LCI assay. Agrobacterium
strains carrying CLuc and NLuc constructs were simply mixed, infiltrated into leaves of N.
benthamiana, and the infiltrated leaves were covered by plastic for 2 days to maintain humidity.
Leaves co-expressing different constructs were then examined for luciferase activity. CLuc-RAR1
was tested for interactions with SGT1a-NLuc. Figure 6 shows that the expression of SGT1a-NLuc
Chen et al., page 13
and the empty 35S::CLuc vector or CLuc-RAR1 construct and the empty 35S::NLuc vector did
not show luciferase complementation, whereas co-infiltration of Agrobacteria containing
CLuc-RAR1 plus SGT1a-NLuc resulted in strong luciferase complementation. The luciferase
activity was ~10 fold greater than the empty vector controls and 7 fold greater than the negative
control expressing SGT1-NLuc and CLuc-CHORD I, indicating a specific interaction. Notably,
the Agrobacterium-based LCI assay was more sensitive and had very low background. We also
determined the time course for luciferase complementation following the co-expression of
SGT1a-NLuc and CLuc-RAR1. Maximum LUC activity was detected 4-6 days after infiltration of
Agrobacterium containing SGT1a-NLuc and CLuc-RAR1, whereas leaves expressing
SGT1a-NLuc and the negative control construct CLuc-CHORD I had only negligible LUC
activity (Figures 7A and 7B). Western blot showed that maximum protein accumulation occurred
between 4-6 days post-infiltration (Figure 7C), indicating that the LUC activity is correlated with
the SGT1a-NLuc and CLuc-RAR1 protein level in the leaves. The CLuc-CHORD I and
CLuc-RAR1 proteins were expressed at a comparable level, indicating that the difference in
luciferase activity was not caused by different amounts of proteins accumulated in the leaves.
Similarly, Agrobacterium-based LCI assay detected specific interaction between SGT1b-NLuc and
CLuc-RAR1 (Figure S3). Although the accumulation of SGT1b-NLuc in leaves was too low to be
detected by western blot, all three negative controls showed only background level of
luminescence that was at least 15 fold less than leaf panels expressing SGT1b-NLuc and
CLuc-RAR1.
DISCCUSION
Chen et al., page 14
In this study we explored the utility of LCI for protein-protein interaction studies in plants. By
using protoplast- and Agrobacterium-based transient expression, we tested the interactions for 9
protein pairs in plants, including components of the SCF E3 ubiquitin ligase complex, HSP90
chaperon complex, bacterial effector-plant resistance protein complex, and transcription factors.
The tested proteins possess a variety of biochemical functions, and the strength of interactions
varies considerably from protein to protein. We observed expected luciferase complementation for
all proteins tested. Importantly, we included strict negative controls for protein-protein interactions,
including unrelated proteins and/or mutant proteins that are specifically compromised in
protein-protein interactions. Whenever possible, the protein level was determined except for one
protein pair. These allowed critical assessment of the detected luciferase complementation,
indicating that LCI is well suited for plant protein-protein interaction studies. In previous studies
in animal systems, the interacting proteins have been successfully positioned to both N-terminal
and C-terminal ends of the fusion construct to achieve complementation (Luker et al., 2004;
Paulmurugan and Gambhir, 2005), suggesting that the luciferase reporter is sufficiently flexible
for different construction strategy.
Non-specific interactions are an inherent problem associated with all protein-protein interaction
assays. In our protoplast-based LCI assays, several negative controls showed certain level of
background signal. It is possible that the two halves of firefly luciferase are capable of association
when present at a high concentration. Nevertheless, the non-specific luciferase activity, as
determined by using mutant or truncated proteins that are known to interfere with protein-protein
interactions, was significantly lower than the positive interactions, indicating that non-specific
Chen et al., page 15
interaction does not impede the proper determination of true interactions. The specificity of
interactions was further enhanced when Agrobacterium-mediated transient expression was used
for LCI. Luciferase activity resulted from specific RAR1-SGT1 interactions were 7-15 times
greater than the negative control (SGT1-NLuc and CHORD I-CLuc) in the Agrobacterium-based
LCI assay, indicating that the Agrobacterium-based transient expression is particularly suited for
protein-protein interaction studies in plants.
Among the methods measuring protein-protein interactions, the yeast two-hybrid method is most
widely used because of the ease of the assay and suitability for large scale screening. However, the
protein-protein interactions are studied in a heterologous system that is prone to false positives. It
is not uncommon that the interaction of two proteins occurs in the presence of additional proteins
or cellular factors. The lack of theses factors in yeast also contributes to false-negatives in yeast
two-hybrid assays. Like FRET and BiFC, LCI detects protein-protein in the native physiological
environment and is thus relevant to biological problems under investigation. Unlike FRET and
BiFC, the current LCI technology does not provide information concerning the subcellular
location of the interaction (Fujikawa and Kato, 2007), a caveat that can be addressed by protein
co-localization analysis.
The LCI assays described in this study have several advantages over FRET and BiFC assays. First,
LCI assays are highly quantitative which allow linear measurement of luminescence signals over
the range of several orders of magnitude. Second, compared to FRET and BiFC, LCI samples the
entire tissue or cell population, avoiding bias derived from individual cells. Third, FRET and BiFC
Chen et al., page 16
assays are complicated by autofluorescence generated by chlorophyll and cell wall. In contrast,
LCI measures luminescence at dark and is not affected by the chlorophyll- and cell wall-generated
autofluorescence. In addition, LCI can be used to study protein-protein interactions at the
organismal level (Luker et al., 2004), and the technique is very useful for studying tissue-specific
protein-protein interactions. Finally, LCI does not require the use of microscope, and data can be
collected within two minutes by using a low light imaging system. This is particularly attractive
for studying the dynamic of protein-protein interactions. The assay can also be done with a
luminometer, so that a large number of protein pairs can be tested simultaneously. The
Agrobaceterium-mediated LCI assay requires minimum sample handling and laboratory training.
This system enables simultaneous test of multiple protein pairs with little effort. The ability to
simultaneously examine a large number of interacting proteins is a prerequisite of protein
interactome construction. Currently, this is done primarily by using the yeast two-hybrid assays
and informatic tools (Piehler, 2005). The availability of LCI as a simple tool for plant
protein-protein interaction studies will facilitate the validation of protein interactome data
collected from the yeast two-hybrid assays.
MATERIALS AND METHODS
Plants
Arabidopsis (Col-0) plants and Nicotiana benthamiana plants were grown in controlled growth
room at 24oC/20oC day/night with 12 h/day light and 70% humidity. Six week old Arabidopsis
plants were used for protoplast isolation. Seven week old N. benthamiana plants were used for
Chen et al., page 17
Agrobacterium-mediated transient expression.
NLuc and CLuc Constructs
A plant gene expression cassette containing the CaMV 35S promoter and rbs terminator was
excised from p35S-FAST (Yiji Xia, Danforth Plant Science Center, St. Louis) and ligated to
pUC19 at EcoRI and HindIII sites, resulting in 35S-pUC19. CLuc and NLuc were PCR-amplified
from CLuc-FKBP and FRB-NLuc (Luker et al., 2004) and ligated into 35S-pUC19, resulting in
35S::NLuc and 35S::CLuc plasmids. Derivative NLuc- and CLuc-fusion constructs were made by
PCR-amplifying the open reading frames of respective genes with primers listed in Table S1,
digested with KpnI and SalI, or KpnI and PstI, and inserted into 35S::Nluc or 35S::Cluc plasmids.
For Agrobacterium-mediate transient expression in N. benthamiana, the expression cassette was
excised from the 35S::NLuc and 35S::CLuc fusion constructs with EcoRI and HindIII and cloned
into pCAMBIA1300 to form pCAMBIA-NLuc and pCAMBIA-CLuc. The constructs were
mobilized into A. tumefaciens strain GV3101.
Upon request, all constructs described in this publication will be made available in a timely
manner for non-commercial research purposes, subject to the requisite permission from any
third-party owners of all or parts of the material. Obtaining any permissions will be the
responsibility of the requestor.
Protoplast Transfection
Chen et al., page 18
Protoplasts were isolated from 6-week-old Col-0 plants according to Sheen
(http://genetics.mgh.harvard.edu/sheenweb/). 2x105 protoplasts were transfected with indicated
constructs, incubated overnight in a 24 well microtiter plate before luciferase activity was
measured.
Agrobacterium-Mediated Transient Expression
Agrobacterium tumefaciens (strain GV3101) bacteria containing indicated constructs were grown
in LB medium at 28℃ overnight, pelleted, and resuspended to 0.3 OD in induction medium
according to Bundock et al. (1995). The culture was then grown in induction medium for 8-12 h.
Bacteria were then washed once with MS medium containing 10 mM N-morpholino
ethanesulfonic acid (MS-MES; pH5.6), and resuspended in MS-MES medium containing 150 µM
acetosyringone to a final concentration of OD600=0.5. Bacterial suspensions were infiltrated into
young but fully expanded leaves of N. benthamiana plants using a needleless syringe. After
infiltration, plants were immediately covered with plastic bags and placed at 23℃ for 48 h before
bag removal. Plants were then incubated at 28℃ with 16 h light/day before the luciferase activity
was measured.
CCD Imaging and Luciferase activity Measurement
1 mM luciferin was added to protoplasts or sprayed onto leaves, and the materials were kept in
dark for 6 minutes to quench the fluorescence. A low light cooled charge-coupled device (CCD)
imaging apparatus (CHEMIPROHT 1300B/LND, 16 bits, Roper Scientific, NJ) was used to
capture luciferase image. The camera was cooled to -110oC and measurement of relative luciferase
Chen et al., page 19
activity as described (He et al., 2004). An exposure time of 2 minutes with 3x3 binning was used
for all images taken. Relative LUC activity is equivalent to luminescence intensity/200 protoplasts
or luminescence intensity/0.2 mm2 leaf area. Each data point consisted of at least 3 replicates, and
3-5 independent experiments were performed for each assay. t test was performed to determine
statistic significance of differences at P<0.01.
Western Blot
Total protein was extracted from equal amounts of protoplasts or leaves, and approximately 100
µg protein was fractionated through SDS Page. Unless indicated otherwise, protein blot was
hybridized with the rabbit anti- full-length firefly luciferase antibodies (Sigma, MO), which react
with both the N-terminal and C-terminal firefly luciferase fragments. The protein blot was
detected with the ECL kit from Amersham Biosciences (NJ). Anti-RAR1 and anti-SGT1
antibodies were raised in-house as previously described (Azevedo et al., 2002).
ACKNOWLEDGEMENTS
We thank David Piwnica-Worms for providing the NLuc and CLuc plasmids and Yan Guo for the
CLuc-ScaBP construct. J.M.Z. was supported by a grant from Chinese Ministry of Science and
Technology (2003-AA210080).
Chen et al., page 20
LITERATURE CITED
Azevedo C, Sadanandom A, Kitagawa K, Freialdenhoven A, Shirasu K, Schulze-Lefert P
(2002) The RAR1 interactor SGT1, an essential component of R gene-triggered disease
resistance. Science 295: 2077–2080.
Bracha-Drori K, Shichrur K, Katz A, Oliva M, Angelovici R, Yalovsky S, Ohad N (2004).
Detection of protein-protein interactions in plants using bimolecular fluorescence
complementation. Plant J 40: 419-427.
Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJ (1995) Trans-kindom T-DNA
transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:
3206–3214.
Callis J, Vierstra RD (2000) Protein degradation in signaling. Curr Opin Plant Biol 3: 381–386.
Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe interactions: shaping the
evolution of the plant immune response. Cell 124: 803-814.
Conti E, Franks NP, Brick P. (1996) Crystal structure of firefly luciferase throws light on a
superfamily of adenylate-forming enzymes. Structure 4: 287-298.
Dixit R, Cyr R, Gilroy S (2006) Using intrinsically fluorescent proteins for plant cell imaging.
Plant J 45: 599-615.
Dong CH, Agarwal M, Zhang Y, Xie Q, Zhu JK (2006) The negative regulator of plant cold
responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of
ICE1. Proc Natl Acad Sci USA 103: 8281-8286.
Fields S, Song OK (1989) A novel genetic system to detect protein–protein interactions. Nature
340: 245–246.
Fujikawa Y, Kato N (2007) Split luciferase complementation assay to study protein-protein
Chen et al., page 21
interactions in Arabidopsis protoplasts. Plant J doi: 10.1111/j.1365-313X.2007.03214.x.
Galarneau A, Primeau M, Trudeau LE, Michnick SW (2002) Beta-lactamase protein fragment
complementation assays as in vivo and in vitro sensors of protein-protein interactions. Nat
Biotechnol 20: 619-622.
Guo H, Ecker JR (2003) Plant responses to ethylene gas are mediated by
SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor. Cell 115: 667-677.
Ha T, Enderle Th, Ogletree DF, Chemla DS, Selvin PR Weiss S (1996) Probing the interaction
between two single molecules: Fluorescence resonance energy transfer between a single
donor and a single acceptor. Proc Natl Acad Sci USA 93: 6264-6268.
Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer
wavelengths and fluorescence resonance energy transfer. Curr Biol 6: 178-182.
He P, Chintamanani S, Chen Z, Zhu L, Kunkel BN, Alfano JR, Tang X, Zhou JM (2004)
Activation of a COI1-dependent pathway in Arabidopsis by Pseudomonas syringae type III
effectors and coronatine. Plant J 37: 589-602.
Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel
family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:
789-798.
Li S, Armstrong CM, Bertin N, Ge H, Milstein S, Boxem M, Vidalain PO, Han JD, Chesneau
A, Hao T (2004) A map of the interactome network of the metazoan C. elegans. Science 303:
540-543.
Chen et al., page 22
Li X, Lin H, Zhang W, Zou Y, Zhang J, Tang X, Zhou JM (2005) Flagellin induces innate
immunity in nonhost interactions that is suppressed by Pseudomonas syringae effectors. Proc
Natl Acad Sci USA 102: 12990-12995.
Luker KE, Smith MC, Luker GD, Gammon ST, Piwnica-Worms H, Piwnica-Worms D (2004)
Kinetics of regulated protein-protein interactions revealed with firefly luciferase
complementation imaging in cells and living animals. Proc Natl Acad Sci USA 101:
12288-12293.
Mahajan NP, Linder K, Berry G, Gordon GW, Heim R (1998) Bcl-2 and Bax interactions in
mitochondria probed with green fluorescent protein and fluorescence resonance energy
transfer. Nature Biotechnology 16: 547 – 552.
Mucyn TS, Clemente A, Andriotis VM, Balmuth AL, Oldroyd GE, Staskawicz BJ, Rathjen
JP (2006) The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate
specific plant immunity. Plant Cell 18: 2792-2806.
Nomura K, DebRoy S, Lee YH, Pumplin N, Jones J, He SY (2006) A bacterial virulence protein
suppresses host immunity to cause plant disease. Science 313: 220-223.
Paulmurugan R, Gambhir SS (2005). Firefly Luciferase Enzyme Fragment Complementation
for Imaging in Cells and Living Animals. Anal Chem 77: 1295-1302.
Piehler J (2005) New methodologies for measuring protein interactions in vivo and in vitro. Curr
Opin Structural Biol 15: 4-14.
Potuschak T, Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C, Genschik P (2003)
EIN3-dependent regulation of plant ethylene hormone signaling by two Arabidopsis F box
proteins: EBF1 and EBF2. Cell 115: 679-689.
Chen et al., page 23
Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Seraphin B
(2001) The tandem affinity purification (TAP) method: a general procedure of protein
complex purification. Methods 24: 218-229.
Quan R, Lin H, Mendoza I, Zhang Y, Cao W, Yang Y, Shang M, Chen S, Pardo JM, Guo Y
(2007) SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to
protect Arabidopsis shoots from salt stress. Plant Cell 19: 1415-1431.
Remy I, Michnick S (1999) Clonal selection and in vivo quantitation of protein interactions with
protein-fragment complementation assays. Proc Natl Acad Sci USA 96: 5394-5399.
Rohila JS, Chen M, Chen S, Chen J, Cerny R, Dardick C, Canlas P, Xu X, Gribskov M,
Kanrar S, Zhu JK, Ronald P, Fromm ME (2006) Protein-protein interactions of tandem
affinity purification-tagged protein kinases in rice. Plant J 46: 1-13.
Rossi F, Charlton CA, Blau HM (1997) Monitoring protein–protein interactions in intact
eukaryotic cells by b-galactosidase complementation. Proc Natl Acad Sci USA 94:
8405–8410.
Shang Y, Li X, Cui H, He P, Thilmony R, Chintamanani S, Zwiesler-Vollick J, Gopalan S,
Tang X, Zhou JM (2006) RAR1, a central player in plant immunity, is targeted by
Pseudomonas syringae effector AvrB. Proc Natl Acad Sci USA 103: 19200-19205.
Shen QH, Saijo Y, Mauch S, Biskup C, Bieri S, Keller B, Seki H, Ulker B, Somssich IE,
Schulze-Lefert P (2007) Nuclear activity of MLA immune receptors links isolate-specific
and basal disease-resistance responses. Science 315: 1098-1103.
Chen et al., page 24
Shirasu K, Lahaye T, Tan MW, Zhou F, Azevedo C, Schulze-Lefert P (1999) A novel class of
eukaryotic zinc-binding proteins is required for disease resistance signaling in barley and
development in C. elegans. Cell 99: 355-366.
Subramanian C, Woo J, Cai X, Xu X, Servick S, Johnson CH, Nebenfuhr A, von Arnim AG
(2006) A suite of tools and application notes for in vivo protein interaction assays using
bioluminescence resonance energy transfer (BRET). Plant J 48: 138-152.
Subramaniam R, Desveaux D, Spickler C, Michnick SW, Brisson N (2001) Direct
visualization of protein interactions in plant cells. Nat Biotechnol 19: 769-772.
Takahashi A, Casais AC, Ichimura K, Shirasu K (2003) HSP90 interacts with RAR1 and SGT1
and is essential for RPS2-mediated disease resistance in Arabidopsis. Proc Natl Acad Sci USA
100: 11777-11782
Tang X, Frederick RD, Zhou J, Halterman DA, Jia Y, Martin GB (1996) Initiation of Plant
Disease Resistance by Physical Interaction of AvrPto and Pto Kinase. Science 274:
2060-2063.
Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C,
Schumacher K, Oecking C, Harter K, Kudla JS3 (2004) Visualization of protein
interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40:
428-438.
Xing W, Zou Y, Liu Q, Liu J, Luo X, Huang Q, She C, Zhu L, Bi R, Hao Q, Wu J-W, Zhou
JM, Chai J (2007) Structural basis for activation of plant immunity by bacterial effector
protein AvrPto. Nature 449: 243-247.
Chen et al., page 25
Xu Y, Piston DW, Johnson CH (1999) A bioluminescence resonance energy transfer (BRET)
system: application to interacting circadian clock proteins. Proc Natl Acad Sci USA 96:
151-156.
Xu L, Liu F, Lechner E, Genschik P, Crosby WL, Ma H, Peng W, Huang D, Xie D (2002) The
SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis.
Plant Cell 14: 1919-1935.
Xu X, Chen C, Fan B, Chen Z (2006) Physical and functional interactions between
pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors.
Plant Cell 18: 1310-1326.
Chen et al., page 26
Figure Legends
Figure 1. Constructs for LCI assays in plants.
A, Schematic diagrams of 35S::NLuc and 35S::CLuc constructs. L denotes the Gly/Ser linker
between LUC fragments and multiple cloning sites (MCS). rbs indicates the transcription
terminator derived from the Rbisco small subunit gene. B, Diagram for luciferase
complementation resulted from an NLuc- and CLuc- fusion proteins.
Figure 2. Interactions of P. syringae effectors with host proteins in protoplasts.
A, Interaction between AvrB and RAR1. B, Interaction between Pto and the N-terminus of Prf. C,
Interaction between AvrPto and Pto. The top panels show quantification of luciferase activity.
Different letters above the bars indicate statistic difference at P<0.01 (t test). The images in the
middle show microtiter plates containing protoplasts expressing the indicated constructs. The
pseudocolor bar below shows the range of luminescence intensity in each image. The bottom
panels show western blot for proteins isolated from protoplasts. Anti-full-length firefly luciferase
antibodies or the indicated specific antibodies (anti-RAR1; Shang et al., 2006; anti-CLuc
antibodies; Sigma) were used to detect the indicated fusion proteins. The amount of protein loaded
in each lane is indicated by Ponceau S staining of Rubisco on a representative protein blot. The
data shown are a representative of 3 independent experiments.
Figure 3. Interactions among the HSP90 chaperone complex components in protoplasts.
A, Interaction between SGT1b and RAR1. B, Interaction between SGT1a and RAR1. C,
Interaction between RAR1 and HSP90. CH I: CHORD I domain; CH II: CHORD II domain; HSP:
Chen et al., page 27
HSP90. The data shown are a representative of 3 (A and B) or 5 (C) independent experiments.
Figure 4. Interaction between WRKY40 and WRKY18.
The WRKY18D construct lacks the leucine zipper motif. The data shown are a representative of 3
independent experiments.
Figure 5. Interactions between ASK1 and COI1. The data shown are a representative of 4
independent experiments.
Figure 6. Interactions between SGT1 and RAR1 in N. benthamiana leaves.
A, Luciferase image of N. benthamiana leaves co-infiltrated with the Agrobacterial strains
containing SGT1a-NLuc and CLuc-RAR1. Arrows indicate leaf panels that were infiltrated with
Agrobacteria containing the indicated constructs. B, Quantification of luciferase activity in leaves
expressing SGT1a-NLuc and CLuc-RAR1. The western blot below shows the expression levels of
CLuc- and NLuc- fusion proteins. RAR1 derivatives were detected by anti-firefly luciferase
antibodies, whereas SGT1a-NLuc was detected by anti-SGT1 antibodies. Ponceau S staining
shows equal loading of protein in lanes. Data were collected 4 days after infiltration. The data
shown are a representative of 3 independent experiments.
Figure 7. Time course of SGT1a-RAR1 interaction in N. benthamiana.
A, Luciferase image of N. benthamiana leaves co-infiltrated with the Agrobacterial strains
containing SGT1a-NLuc and CLuc-RAR1. B, Quantification of luciferase activity in leaves
Chen et al., page 28
expressing SGT1a-NLuc and CLuc-RAR1. C, Accumulation of fusion proteins in N. benthamiana
leaves. Data were collected at the indicated days post infiltration of Agrobacterium. Mock: leaves
infiltrated with H2O. Arrows indicate infiltrated region. The data shown are a representative of 5
independent experiments.